Transient Recombinant Protein Production in Glycoengineered Nicotiana benthamiana Cell Suspension Culture.

Sara Sukenik,Carlito Lebrilla,Qiongyu Li,Somen Nandi,Kalimuthu Karuppanan,Karen Mcdonald

doi:10.3390/ijms19041205

Sara Sukenik, Carlito Lebrilla + Show 4 more

Open Access

https://doi.org/10.3390/ijms19041205

Copy DOI

Abstract

Transient recombinant protein production is a promising alternative to stable transgenic systems, particularly for emergency situations in which rapid production of novel therapeutics is needed. In plants, Agrobacterium tumefaciens can be used as a gene delivery vector for transient expression. A potential barrier for plant-based production of human therapeutics is that different glycosylation patterns are found on plant and mammalian proteins. Since glycosylation can affect the efficacy, safety and stability of a therapeutic protein, methods to control glycan structures and distributions in plant-based systems would be beneficial. In these studies, we performed Agrobacterium-mediated transient expression in glycoengineered plant cell suspension cultures. To reduce the presence of plant-specific glycans on the product, we generated and characterized cell suspension cultures from β-1,2-xylosyltransferase and α-1,3-fucosyltransferase knockdown Nicotiana benthamiana. An anthrax decoy fusion protein was transiently produced in these glycoengineered plant cell suspension cultures through co-culture with genetically engineered Agrobacterium. The mass ratio of Agrobacterium to plant cells used was shown to impact recombinant protein expression levels. N-glycosylation analysis on the anthrax decoy fusion protein produced in glycoengineered N. benthamiana showed a dramatic reduction in plant-specific N-glycans. Overall, the results presented here demonstrate the feasibility of a simple, rapid and scalable process for transient production of recombinant proteins without plant-specific glycans in a glycoengineered plant cell culture host.

Highlights

Rapid, large-scale production of novel drugs or vaccines would be invaluable in emergency situations, such as an infectious disease outbreak or bioterrorist attack
The ∆XTFT N. benthamiana callus line was generated from aseptically grown plants (Figure 1A) by culturing explants on semi-solid media (Figure 1B)
It is hypothesized that the plant cell growth phase impacts the expression level obtained for any given mass ratio of Agrobacterium added

Summary

Introduction

Large-scale production of novel drugs or vaccines would be invaluable in emergency situations, such as an infectious disease outbreak or bioterrorist attack. While agroinfiltration can produce recombinant proteins with short lead times, limitations of a whole plant transient expression system include the need to grow and manipulate whole plants and challenges associated with purification of the product from plant biomass. We are investigating Agrobacterium-mediated transient expression in plant cell suspension cultures as an alternative production system. A transient cell culture system would have short lead times yet could allow controlled and optimized environmental conditions in a bioreactor for plant growth and protein production while simplifying purification by targeting the protein for secretion. Plant cell packs have been used for transient expression [9] In this system, a porous cell pack is created using vacuum filtration to remove the culture media. Similar to whole plant processes, a cell pack process would require specialized commercial manufacturing facilities

Results

Discussion

Conclusion